This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. So far, cryo-EM reconstruction of single particles determined at subnanometer resolution have been readily, convincingly and successfully applied to relatively large nanomachines. It is known that many biological processes are carried out by macromolecular complexes which have a molecular mass between 50-300 kDa. Such relatively small sizes pose a challenge for single particle reconstruction because these complexes are often difficult to localize under typical cryo-EM conditions, thus making it difficult or impossible to determine the particles? orientations. We are exploring the use of a heated phase plate to record images. Nagayama and colleagues in Japan have demonstrated images with strikingly high contrast for a number of biological specimens ranging from single particles to whole cells. Their most recent work using a heated phase plate positioned in the focal plane of an objective lens having a longer focal length allowed them to image GroEL and reconstruct it to 12 [unreadable] resolution (Danev and Nagayama, 2008). Our goal in this project is to explore the use of such imaging technology.